Automated Bright Field Segmentation of Cells and Vacuoles Using Image Processing Technique

Understanding the mechanisms and other variants of programmed cell death will help provide deeper insight into various disease processes. Although complex procedures are required to distinguish each type of cell death, the formation of vacuoles is one of the important features in some process of cell death under different conditions. Thus, monitoring and counting the number of vacuoles and the ratio of cells with vacuoles is a commonly used method to indicate and quantify the efficacy of the therapy. Several studies have shown that image processing can provide a quick, convenient and precise mean of performing cell detection. Hence, this study uses an image processing technique to detect and quantify vacuolated cells without the need for dyes. The system both counts the number of vacuolated cells and determines the ratio of cells with vacuoles. The performance of the proposed image processing system was evaluated using 38 images. It has been shown that a strong correlation exists between the automated counts and the manual counts. Furthermore, the absolute percentage errors between automated counts and manual counts for cell detection and vacuolated cell detection using data pooled from all images are 3.61 and 3.33%, respectively. A user‐friendly graphical user interface (GUI) is also developed and freely available for download, providing researchers in biomedicine with a more convenient instrument for vacuolization analysis.

[1]  Olli Yli-Harja,et al.  Software for quantification of labeled bacteria from digital microscope images by automated image analysis. , 2005, BioTechniques.

[2]  Min Li,et al.  A Novel Segmentation Method for Overlapping Cells , 2006, 2006 6th World Congress on Intelligent Control and Automation.

[3]  K. Ryan,et al.  The multiple roles of autophagy in cancer , 2011, Carcinogenesis.

[4]  Per O. Seglen,et al.  Methods for Monitoring Autophagy from Yeast to Human , 2007, Autophagy.

[5]  John L Cleveland,et al.  Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes , 2008, Autophagy.

[6]  Fabian Rudolf,et al.  Accurate cell segmentation in microscopy images using membrane patterns , 2014, Bioinform..

[7]  Amanda Capes-Davis,et al.  Check your cultures! A list of cross‐contaminated or misidentified cell lines , 2010, International journal of cancer.

[8]  W. Chang,et al.  Targeting cathepsin S induces tumor cell autophagy via the EGFR-ERK signaling pathway. , 2012, Cancer letters.

[9]  Luc Vincent,et al.  Watersheds in Digital Spaces: An Efficient Algorithm Based on Immersion Simulations , 1991, IEEE Trans. Pattern Anal. Mach. Intell..

[10]  Kevin A Roth,et al.  Chloroquine-induced autophagic vacuole accumulation and cell death in glioma cells is p53 independent. , 2010, Neuro-oncology.

[11]  F. P. León,et al.  Suppression of inhomogeneities in images of textured surfaces , 1997 .

[12]  S. Elmore.  Apoptosis: A Review of Programmed Cell Death , 2007, Toxicologic pathology.

[13]  T. He,et al.  Paraptosis and NF-κB activation are associated with protopanaxadiol-induced cancer chemoprevention , 2013, BMC Complementary and Alternative Medicine.

[14]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[15]  S. Spiegl-Kreinecker,et al.  Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity , 2013, Cell Death and Disease.

[16]  Stephen V. Stehman,et al.  Selecting and interpreting measures of thematic classification accuracy , 1997 .

[17]  Andrew G. Dempster,et al.  Analysis of infected blood cell images using morphological operators , 2002, Image Vis. Comput..

[18]  Justin W. L. Wan,et al.  A combined watershed and level set method for segmentation of brightfield cell images , 2009, Medical Imaging.

[19]  H. Vihinen,et al.  Monitoring autophagy by electron microscopy in Mammalian cells. , 2009, Methods in enzymology.

[20]  E. Eskelinen.  Fine structure of the autophagosome. , 2008, Methods in molecular biology.

[21]  A. Ciechanover,et al.  p62- and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome , 2016, Proceedings of the National Academy of Sciences.

[22]  Yongqiang Chen,et al.  Is mitochondrial generation of reactive oxygen species a trigger for autophagy? , 2008, Autophagy.

[23]  Takeo Kanade,et al.  Cell population tracking and lineage construction with spatiotemporal context , 2008, Medical Image Anal..

[24]  Justin W.L. Wan,et al.  A spectral k-means approach to bright-field cell image segmentation , 2010, 2010 Annual International Conference of the IEEE Engineering in Medicine and Biology.

[25]  Daniel J. Klionsky,et al.  Autophagy in Health and Disease: A Double-Edged Sword , 2004, Science.

[26]  Matthew J. Simpson,et al.  Sensitivity of Edge Detection Methods for Quantifying Cell Migration Assays , 2013, PloS one.

[27]  Yongqiang Chen,et al.  Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells , 2008, Cell Death and Differentiation.

[28]  Michael Brady,et al.  Advanced phase-based segmentation of multiple cells from brightfield microscopy images , 2008, 2008 5th IEEE International Symposium on Biomedical Imaging: From Nano to Macro.

[29]  T. Lamark,et al.  Regulation of selective autophagy: the p62/SQSTM1 paradigm. , 2017, Essays in biochemistry.

[30]  Jens Rittscher,et al.  Spatio-temporal cell cycle phase analysis using level sets and fast marching methods , 2009, Medical Image Anal..

[31]  William A Weiss,et al.  Principles and Current Strategies for Targeting Autophagy for Cancer Treatment , 2011, Clinical Cancer Research.

[32]  N. Mizushima.  Methods for monitoring autophagy. , 2004, The international journal of biochemistry & cell biology.

[33]  G. Bjørkøy,et al.  Monitoring autophagic degradation of p62/SQSTM1. , 2009, Methods in enzymology.

[34]  T. He,et al.  Ginsenoside Rh2 induces apoptosis and paraptosis-like cell death in colorectal cancer cells through activation of p53. , 2011, Cancer letters.

[35]  Michael Brady,et al.  PHASE-BASED SEGMENTATION OF CELLS FROM BRIGHTFIELD MICROSCOPY , 2007, 2007 4th IEEE International Symposium on Biomedical Imaging: From Nano to Macro.

[36]  N. Mizushima,et al.  How to Interpret LC3 Immunoblotting , 2007, Autophagy.

[37]  Heng Tao Shen,et al.  Principal Component Analysis , 2009, Encyclopedia of Biometrics.

[38]  Jack Sklansky,et al.  Minimum-Perimeter Polygons of Digitized Silhouettes , 1972, IEEE Transactions on Computers.

[39]  Franz Kummert,et al.  Automatic Segmentation of Unstained Living Cells in Bright-Field Microscope Images , 2006, Industrial Conference on Data Mining - Workshops.

[40]  Hans De Sterck,et al.  Multilevel Space-Time Aggregation for Bright Field Cell Microscopy Segmentation and Tracking , 2010, Int. J. Biomed. Imaging.

[41]  Yongqiang Chen,et al.  Superoxide is the major reactive oxygen species regulating autophagy , 2009, Cell Death and Differentiation.

[42]  Kenong Wu,et al.  Live cell image segmentation , 1995, IEEE Transactions on Biomedical Engineering.

[43]  S. Barth,et al.  Autophagy: assays and artifacts , 2010, The Journal of pathology.

[44]  Erik H. W. Meijering,et al.  Cell Segmentation: 50 Years Down the Road [Life Sciences] , 2012, IEEE Signal Processing Magazine.

[45]  Yongqiang Chen,et al.  Mitochondrial electron-transport-chain inhibitors of complexes I and II induce autophagic cell death mediated by reactive oxygen species , 2007, Journal of Cell Science.

[46]  C. Pestana,et al.  Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry Inhibition of Autophagy by Chloroquine Stimulates Nitric Oxide Production and Protects Endothelial Function during Serum Deprivation , 2022 .

[47]  Daniel J. Klionsky,et al.  Autophagy: from phenomenology to molecular understanding in less than a decade , 2007, Nature Reviews Molecular Cell Biology.

[48]  A. H. Mir,et al.  Segmentation of living cells: A comparative study , 2014, 2014 International Conference on Communication and Signal Processing.

[49]  Yongqiang Chen,et al.  Methods for detecting autophagy and determining autophagy-induced cell death. , 2010, Canadian journal of physiology and pharmacology.